Lipid Profile and Apolipoprotein E Genotyping in Stroke:

A Case-Control Study

Mustafa Serteser¹, Sophie Viskikis², Tomris Ozben³, Bernard Herbeth², Sevin Balkan⁴, Gerard Siest²

¹Afyon Kocatepe University, School of Medicine, Department of Biochemistry, Inonu Bulvari,03200, Afyon, Turkey,²Centre de Medecine Preventive Upes-Interaction-Gene-Environment, Universite Henri Poincare, Nancy I, 2, Avenue du Doyen Jacques Parisot 54501, Vandoeuvre-Les-Nancy, France, Akdeniz University School of Medicine Department of Biochemistry³ and Neurology⁴, 07070, Antalya Turkey

Corresponding Author:

Mustafa SERTESER M.D.

Afyon Kocatepe University, School of Medicine, Department of Biochemistry, Inonu Bulvari,03200, Afyon, Turkey

serteser@aku.edu.tr

ABSTRACT

The possible effect of the apolipoprotein E polymorphism on the development of ischemic cerebrovascular disease has not been sufficiently investigated, and controversial results were obtained from the few existing studies. In this study, our goal was to determine the possible role of the apolipoprotein E polymorphism in stroke patients. Genotyping of apolipoprotein E carried out on 79 patients (26 thrombotic, 20 embolic, 26 lacunar, and 7 miscellaneous), and in 126 age and sex matched controls who were free of cerebrovascular disease. In addition, serum apolipoprotein E, A I, C III and B and lipoprotein (a) levels were determined. The prevalence of well-known vascular risk factors was significantly higher in the patients. The e2 allele was found to be significantly lower in patients (3.16 %) than in controls (8.34%) (c² = 4.37, p<0.05). Patients with large-vessel stroke or lacunar stroke had higher triglyceride and lower HDL levels. In all stroke subtypes, apolipoprotein A I levels were lower than those in controls, and the ratio of apolipoprotein B to A I was higher. A stepwise logistic regression showed that; the presence of vascular stroke was related to e4 allele, diabetes, high systolic blood pressure, and high apolipoprotein E serum levels, but inversely related to e2 allele and apolipoprotein A I levels. Epsilon 2 may protect individuals against stroke even if the e2 carrying patients have higher apo E levels, and e4 may be a genetic risk factor together with other well-known vascular risk factors. Large population-based studies are needed to clarify the exact relationship between stroke and lipid metabolism.

KEY WORDS:

Stroke, apo E, polymorphism

INTRODUCTION

Stroke is the third major cause of death and long term disability in industrialized countries. Intracerebral and subarachnoid haemorrhages account for only 15 % of all strokes whereas the other 85 % are caused by cerebral ischemia and can be distinguished according to the cause, clinical syndrome or the arterial distribution. Stroke due to large artery atherosclerosis causes infarcts, which are larger than 1.5 cm along with the territory of the major intracerebral arteries. Cardiac embolism also gives a similar clinical picture to large-artery atherosclerosis. In small artery occlusion, which is caused by lipohyalinosis of small perforating vessels, small brain stem or subcortical lesions of less than 1.5 cm are detected. The other rare causes of strokes are vasculitis, hemotologic disorders, migraine or oral contraceptives (van Gijn and van der Worp,1995).Cerebrovascular diseases are not only induced by classic vascular risk factors including hypertension, diabetes mellitus, cigarette smoking, but also by genetic factors (Pullicino et al.,1997).

Previously, the role of apolipoprotein E (apo E) polymorphism in atherosclerotic events has been shown (Utermann,1987). Apo E is a protein which acts as a ligand for low density lipoprotein (LDL) receptors and affects the hepatic binding, uptake and catabolism of different lipoproteins. It also has a function in the repair response to tissue injury. Increased levels of apo E concentration have been shown at sites of peripheral nerve injury and regeneration (Mahley,1988). It has been found that the gene for apo E is located on chromosome 19. Six major isoforms of the apo E gene exist, each contains a pair among three major alleles e2, e3, e4 which encode the protein isoforms E2, E3, E4 (Siest et al.,1995; Mahley,1988). Clinical and postmortem studies have shown that the e4 allele is associated with pathologies such as coronary artery diseases or Alzheimer's disease (Kosunen et al.,1995; Strittmatter and Roses, 1995, Wang et al.,1995; Corder et al.,1993;Saunders et al.,1993). In patients with ischemic cerebrovascular diseases (ICVD), only a limited number of studies clarifying the role of apolipoproteins and the role of genetic polymorphisms in lipoprotein metabolism have been performed. Studies searching for the effect of the apo E polymorphism are also rare and have produced contradictory results (Ferruci et al.,1997; Kessler et al.,1997; Schmidt et al.,1997; Kuusisto et al.,1995; Couderc et al.,1993; Pedro-Botet et al.,1992).

In this study, we aimed to find out the relationship, if one exists,between ICVD, lipids, apolipoprotein levels and apo E polymorphism. Moreover we searched for a possible relationship between apo E polymorphism and subtypes of stroke.

MATERIALS AND METHODS

Patient selection:

The study group consisted of patients admitted to the Neurology Department of Akdeniz University Hospital and to the other two major hospitals in the city, who had suffered from an ischemic event or patients admitted to the Neurology Out-patient Clinics for follow-up, who had previously been diagnosed as stroke patients within the last 15 months. Each patient underwent a complete physical and neurological examination by a neurologist. The control subjects were free of cerebrovascular diseases and were matched with the patients for sex and age on the basis of age classes ( 40-49, 50-59, 60-69, 70-80 ).

Nearly all the patients underwent computerized tomography (CT) or magnetic resonance imaging (MRI) and electrocardiography (ECG) analysis. On the basis of clinical symptoms and findings of diagnostic tools, the patients were assigned into one of the following categories: (1)- Large vessel disease-strokes due to pathologies of extracranial arteries either thrombotic or embolic, (2)- Lacunar stroke due to small deep infarctions in the territory of small perforating arteries of the brain where localization of these infarcts was confirmed by CT or MRI and the matched clinical symptoms, (3) Stroke due to miscellaneous causes which includes cardiac emboli or unknown pathomechanisms. The consciousness level (based on the Glasgow Coma Scale), functional status (based on the Barthel Index) and the level of disability (based on the Rankin Scale) were also determined in patients.

Potential confounders:

Vascular risk factors and associated vascular diseases, based on the individual's personal history, results of a physical examination and appropriate laboratory findings were recorded for patients and controls. Those included; hypertension, diabetes mellitus (DM), tobacco and alcohol consumption, use of oral contraceptives, body mass index (BMI), history of migrane, ischemic heart diseases, arrhythmias, family histories of hypertension, diabetes mellitus and stroke. Arterial hypertension was considered to be present if an individual had a history of hypertension or was using antihypertensive agents or if the systolic blood pressure (SBP) exceeded 140 mmHg or the diastolic blood pressure (DBP) exceeded 90 mmHg. DM was considered to be present if fasting glucose levels were exceeded 7.78 mmol/L or if the individual was using antidiabetic agents.

We only included patients with neurologic symptoms resulting from focal cerebral ischemia and excluded patients with intracerebral or subarachnoid haemorrhage. Patients with stroke resulting from vasculitis, migraine, oral contraceptive use or from trauma were also excluded.

Apo E genotyping:

DNA was prepared from whole blood (Miller et al.,1988). For genotyping of common apoE isoforms, amplification of apoE sequences was carried out (Hixon and Vernier,1990). Electrophoresis of the samples was performed on 10% polyacrylamid gel, after digesting the amplified products with the HhaI restriction enzyme. The detection of restriction fragments was performed by staining with ethidium bromide under UV light.

Determination of parameters of lipid metabolism:

Serum apo E concentrations were determined immunoturbidimetrically by using a kit supplied by Daicchii Pure Chemicals Co., Ltd., Tokyo, Japan. Total cholesterol, HDL-cholesterol (after precipitation procedure with phosphotungustic acid and Mg⁺² ions) and triglyceride concentrations were determined enzymatically by using CHOD/PAP and GPO/PAP methods respectively and the calculation of LDL-cholesterol was performed by using Friedewald formula. Apo AI, apo B and lipoprotein (a) [Lp(a)] levels were determined in a Behring Nephelometer. Quantification of total apo E and total apo C III particles and apo E present in the particles without apo B (apo E LP non B) and apo C III present in particles without apo B (apo C III LP non B) was carried out by an electroimmunodiffusion technique in an agarose gel supplied by Sebia (Issy-les-Moulineaux, France).

Statistical analysis:

Standard statistical procedures from the BMDP statistical software were used. For major risk factors and potential confounders, differences between case and control groups were tested by using Student's t test, ANOVA, Dunnett's test and c² analysis. As the distribution of triglyceride and Lp(a) levels were skewed, the log transformed values were used. To estimate the effect of the apo E polymorphism on the risk of stroke, whilst simultaneously adjusting for possible confounders, unconditional multiple logistic regression was used with maximum likelihood estimation of the regression coefficients and their standard errors. All the potential confounding factors were systematically tested in the regression models: sex, hypertension, diabetes, cigarette and alcohol consumption, family history of stroke and hypertension (as categorical variables), and age, SBP and DBP, BMI, cholesterol, triglyceride, apolipoprotein and lipoprotein levels (as contunious variables). The fitted model included DM, SBP and serum apo E and apo AI levels as confounders. Adjusted odds ratios for stroke were calculated for e2 allele [(e2/ e2) + (e3/ e2) + (e2/ e4)] and for e4 allele [(e3/ e4) + (e4/ e4)] by taking the e3 [(e3/ e3)] allele as a reference. Significance levels were set at 0.05 in all cases.

RESULTS

We studied 79 patients ( mean-/+SD age, 62.9-/+8.9 years, range 40-80 years, 50.6% male) and 126 controls (mean-/+SD age, 58.6+/-8.81 years, range 40-77 years, 47.6% male). The prevalence of genotypes which are shown in Table 1a. and the allele frequencies are shown in Table 1b.

Table 1a: Apo E genotypes in controls and patients

Table 1b: Apo E allelic frequency (Miscellaneous group contains patients with stroke due to cardiac embolism and with unknown cause)

 *p<0,05 Difference between controls and patients (Dunnettis test)

 No statistically significant difference was found between patients and controls for e3 and e4 allele frequencies. But e2 was found to be much more prominent in controls than in patients (c² =4.37, p<0.05). The general characteristics of the patients and controls are seen in Table 2.

Table 2: General characteristics of controls and patients.

 **p<0,05, ***p<0,001: Difference between controls and patients (Student t test or c² test)

No statistically significant sex differences were found between patients and controls but the mean age of patients was higher than those of controls (p<0.05). SBP and DBP were found to be higher in patients than in controls (p<0.001) especially in older individuals. Of the patients, 79% were found to be hypertensive and 28.4% to be diabetic. No significant difference was found between patients and controls for the family history of hypertension and DM, but on the other hand 49.5% of the patients had a positive family history for stroke, compared to 23.8% of the controls (c²=13.40, p<0.05). Current cigarette and alcohol consumption was found to be much more prominent in patients than in controls (c² =20.72, p<0.001, c² =10.02, p<0.05 respectively).

Table 3: Plasma lipid profile in controls and patients according to stroke subtypes. (Miscellaneous group contains patients with stroke due to cardiac embolism and with unknown cause)